The chemistry and bioactivity of phallotoxins, which are one of the main groups of fungal toxins isolated from poisonous mushroom Amanita phalloides, have been studied systematically for over 100 years (For review, see: Wieland, T. Peptides of Poisonous Amanita mushrooms; Springer-Verlag: New York, 1986). The bicyclic phallotoxins are cross-linked by a thioether between the side chains of tryptophan and cysteine residues. The tryptophan residue is substituted by a sulfur group in the 2-position of the indole ring, which is named trypthionine. The phallotoxins bind strongly to filamentous actin (F-actin), not to its monomeric form, G-actin. Actin is a collective name for a class of proteins of about 43 kD, which has been detected as a type of cytoskeletal protein and isolated from many sources. The toxins accelerate the polymerization of G-actin and stabilize F-actin, thus disturbing the F-actin and G-actin equilibrium of this cytoskeletal protein. The bioactivity of the toxins depends on the molecular shape that is critical for toxins' binding to the target proteins. Any change of the molecular conformation, such as removing the sulfur-containing bridges or splitting the peptide bonds, results in the loss of toxicity.
Phallotoxins, such as phalloidin, phallacidin and phalloin are bicyclic heptapeptides that differ by the amino acid residues in the peptides. The structure of the selected phallotoxins is shown in Formula 1.
Formula 1. The general formula of the phallotoxins R1R2R3PhalloidinCH3CH3OHPhallacidinCH(CH3)2COOHOHPhalloinCH3CH3H
All three peptides contain a thioether bridge linking L-Cysteine (Cys3) and L-Tryptophan (Trp6), a cis epimer of 4-hydroxy-L-Proline (cis-Hyp4), and an L-Alanine (Ala5). At position 1 and 2, both phalloidin and phalloin have an L-Alanine (Ala1) and a D-Threonine (DThr2), while phallacidin has an L-Valine (Val1) and a β-hydroxy-D-Asparatic acid (DAsp2). At position 7, both of phalloidin and phallacidin contain an unusual γ,δ-dihydroxy-L-Leucine (γ,δ-di-OH-Leu7), while phalloin has a γ-hydroxy-L-Leucine (γ-OH-Leu7).
The fluorescent phallotoxins used as probes for actin were introduced in 1979 (Wulf, E.; Deboben, A.; Bautz, F. A.; Faulstich, H.; Wieland, T. Proc. Natl. Acad. Sci. USA 1979, 76, 4498-4502) after the first fluorescent phallotoxin was synthesized from the reaction of fluorescein-isothiocyanate with amino-functionalized derivative of ketophalloidin (Wieland, T.; Deboben, A.; Faulstich, H. Liebigs Ann. Chem. 1980, 416-424). Since then the fluorescent phallotoxins have been widely applied in biological research, especially in histological applications. For example, the fluorescent phallotoxins have been used for the visualization of F-actin fibers by staining a variety of cells, which provide a convenient method for labeling, identifying, and quantifying F-actin in muscle and non-muscle cells from different species of plants and animals. (More examples, see: Faulstich, H,; Zobeley, S.; Rinnerthaler, G.; Small, J. V. J. Muscle Res. Cell Motility, 1988, 9, 370-383.; Szczesna, D.; Lehrer, S. S. J. Muscle Res. Cell Motility, 1993, 14, 594-597.; Prochniewicz-Nakayama, E,; Yanagida, T.; Oosawa, F. J. Cell Bio. 1983, 97, 1663-1667.; Small, J.; Zobeley, S.; Rinnerthaler, G.; Faulstich, H. J Cell Sci., 1988, 89, 21-24.; Ao, X.; Lehrer, S. S. J. Cell Sci., 1995, 108, 3397-3403.; Wang, K.; Feramisco, J. R.; Ash, J. F. Methods Enzymol., 1982, 85,514-562.; Adams, A. E. M.; Pringle, J. R. Methods Enzymol., 1991, 194, 729-731.; Schmit, A. C.; Lambert, A. M. The Plant Cell, 1990, 2, 129-138.; Mahaffy, R. E.; Pollard, T. D. Biochemistry, 2008, 47, 6460-6467.; Li, K.; Pu, K. Y.; Cai, L.; Liu, B. Chem. Mater. 2011, 23, 2113-2119.; An, M.; Wijesinghe, D.; Andreev, O. A.; Reshetnyak, Y. K.; Engelnian, D. M. Proc. Natl. Acad. Sci. USA 2010, 107, 20246-20250.
Since the structures of phallotoxins have been recognized, a substantial amount of synthetic work on natural and non-natural analogues of these bicyclic peptides has been carried out, especially in Wieland's laboratory (Wieland, T. Peptides of Poisonous Amanita mushrooms; Springer-Verlag: New York, 1986). Based on the structure-bioactivity relationship studies of phallotoxins, the interaction of phallotoxins with the target protein, F-actin, occurs at the left side 15-membered ring of the molecule (Formula 1), which contains amino acid residues at position 3, 4, 5, 6. Side chains at positions 1 and 2 play a minor role, while the role of side chain at 7 is completely insignificant.
Some functional groups, such as the amino group, are introduced to the side chain-7 and provide a convenient route to attach the fluorophores. For example, [D-Abu2-Lys7]-phalloin has been made in solution-phase peptide synthesis and its rhodamine conjugate was obtained by reaction of the lysine analog with tetramethyl-rhodamine isothiocyanate (Wieland, T.; Miura, T.; Seeliger, A. Int. J. Pept. Protein Res. 1983, 21, 3-10). More recently, [Glu7]-phalloidin was synthesized by a solid-phase peptide synthesis route. The glutamic acid is introduced to the cyclic peptide both as a handle for linkage to resins and as a reactive site for conjugating the tetramethylrhodamine cadaverine (Schuresko, L. A.; Lokey, R. S. Angew. Chem. Int. Ed., 2007, 46, 3547-3549).
The synthesis of the phalloitoxin peptides comprises: (1) the normal coupling of amino acids for generation of the peptide chains, (2) the formation of thioether linkage between tryptophan and cysteine, and (3) the cyclization of the linear peptides by intramolecular head and tail coupling. Based on the key step, the formation of trypthionine, the following routes are used for synthesis of phalloidin derivatives.
Route 1: The thiol of the cysteine residue in one peptide fragment is converted to the corresponding sulfenyl chloride. The S-chloride then reacts with the indole of the tryptophane residue in another peptide fragment to form the trypthionine moiety. The following two cyclization reactions of the double peptide give the bicyclic peptide.
For example, see: Fahrenholz, F.; Faulstich, H.; Wieland, T. Liebigs Ann. Chem., 1971, 743, 58-61; Munekata, E.; Faulstich, H.; Wieland, T. Liebigs Ann. Chem., 1977, 1758-1765; Wieland, T.; Jochum, C.; Faulstich, H. Liebigs Ann. Chem., 1969, 727,138-142.
Route 2: L-3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole-2-carboxylic acid (Hpi), oxidation product of L-tryptophan by peroxy acid, reacts with thiols under acidic condition to yield 2-thioethers of L-tryptophan (Saviage-Fontana reaction, see: Saviage, W. E.; Fontana, A. Int. J. Pept. Protein Res. 1980, 15, 102-112.; J. Chem. Soc., Chem. Comm., 1976, 600-601.; Aust. J. Chem., 1975, 28, 2275-2278). The linear peptides containing a cysteine residue and Boc-protected Hpi are synthesized by a solution-phase peptide synthesis method. The first cyclization forms the intramolecular indolyl-thioethers under the Savige-Fontana reaction conditions, the second cyclization gives the final phalloidin derivatives by intramolecular head-tail coupling.
For example, see: Wieland, T.; Miura, T.; Seeliger, A. Int. J. Pept. Protein Res. 1983, 21, 3-10; Zanotti, G.; Falcigno, L.; Saviano, M.; D'Auria, G.; Bruno, B. M.; Campanile, T.; Paollilo, L. Chem. Eur. J. 2001, 7, 1479-1485; Falcigno, L.; Costantini, S.; D'Auria, G.; Bruno, B. M.; Zobeley, S.; Zanotti, G. Paollilo, L. Chem. Eur. J. 2001, 7, 4665-4673.
Route 3: The cyclic peptide containing tryptophan and S-tritylcysteine is oxidized by iodine to form tryptophan-2-thioether. The intermediate is sulfenyl iodide from the reaction of S-trityl group with iodine. Under some conditions, such as in dilute solution, the further reaction of sulfenyl iodide with S-trityl group to form dimeric disulfide is suppressed and the intramolecular reaction with the indole of tryptophan to form thioether is favored (Sieber, P.; Kamber, B.; Riniker, B.; Rittel, W. Helv. Chim. Acta, 1980, 63, 2358-2363). Recently, by using this strategy, Glu7-phalloidin was synthesized using solid-phase synthesis (Schuresko, L. A.; Lokey, R. S. Angew. Chem. Int. Ed., 2007, 46, 3547-3549.; US patent, 2011, U.S. Pat. No. 7,964,702).

The existing synthetic routes successfully make a number of phallotoxin derivatives. However, each of these routes has drawbacks, such as low yields resulting from multi-step solution-phase peptide synthesis or time-consuming synthesis of the key intermediate. The previous synthesis of phallotoxins and their analogues mainly uses large scale solution-phase peptide synthesis techniques, especially through route 1 and route 2. A solid-phase synthetic approach to Ala7-phalloidin has been developed (Anderson, M.; Shelat, A. A.; Guy, R. K. J. Org. Chem., 2005, 70, 4578-4584). The key intermediates, such as protected cis-Hyp and thioether linked Trp6-Cys3 unit, are still prepared in solution. This method contains two sequential resin-bound macro-cyclization reactions. The second reaction is sluggish and results in the low overall yield due to the formation of oligomers from the side reaction.
In the procedure of solid-phase synthesis of Glu7-phalloidin through route 3, the direct thionation of the indole of tryptophan in the solid phase produces a high yield of the final product (Schuresko, L. A.; Lokey, R. S. Angew. Chem. Int. Ed, 2007, 46, 3547-3549). However, this method is more expensive to perform for large scale synthesis because of the low resin loading that is necessary for successful on-resin cyclizations. Due to the vital importance of phallotoxin derivatives, any conceptually new and practical method for the synthesis of these compounds is of special significance. In this invention, we disclose a new method to make novel functionalized phalloidin derivatives and their fluorescent dye conjugates.